The Ca2+-activated K+ channel, KCa3.1, is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. Our studies on the regulation of KCa3.1 have uncovered a completely new signaling pathway required for the reactivation of human CD4 T cells. This pathway includes a potentially novel phosphatidylinositol 3 Kinase (PI3K) isoform, phosphatidylinositol 3-Phosphate (PI(3)P), and a mammalian histidine kinase, Nucleoside Diphosphate Kinase B (NDPK-B), that are required for KCa3.1 channel activation. We found that NDPK-B functions downstream of PI(3)P to activate KCa3.1 by phosphorylating H358 in KCa3.1's carboxyterminal (CT) tail. Moreover, we have identified 2 new negative regulators of T cell activation, the PI(3)P phosphatase myotubularin related protein 6 (MTMR6) and the histidine phosphatase, phosphohistidine phosphatase-1 (PHPT-1), which inhibit KCa3.1 by dephosphorylating PI(3)P and KCa3.1 respectively. The major goal of this proposal is to further explore the mechanisms whereby these molecules regulate KCa3.1 and their biological relevance to T cell activation and disease. Specific Aim (SA) 1 will identify the signaling pathway in CD4 T cells that is critical for generating the PI(3)P pool that mediates KCa3.1 channel activation and the mechanism whereby PI(3)P functions upstream to regulate NDPK-B. SA2 will determine whether T cell receptor (TCR) stimulation regulates the redistribution of NDPK-B, KCa3.1, MTMR6, and the PI3K isoform identified in SA1 to the immunological synapse (IS), and the consequences this plays in the regulation of KCa3.1 channel activity. SA3 will identify the histidine phosphatase that negatively regulates KCa3.1 channel activity and its role in T cell biology. Our recent data indicates that the histidine phosphatases PHPT-1 and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) are negative regulators of KCa3.1 and function to reverse the activation of KCa3.1 by NDPK-B by dephosphorylating histidine 358 (H358) in the CT of KCa3.1.The Ca2+-activated K+ channel, KCa3.1, is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. Public Health Relevance: Our studies on the regulation of KCa3.1 have uncovered new signaling pathways that functions as both positive and negative regulators of KCa3.1 channel activity. Interfering pharmacologically with molecules we found that positively regulate KCa3.1 may identify novel drug targets to treat transplant rejection and autoimmune disease. On the flip side, abnormal expression or activity of molecules we identified that negative regulate KCa3.1 could provide new insight into autoimmune diseases.